The present invention generally relates to methods for identifying pharmaceutically relevant compounds, and particularly to methods for selecting compounds capable of modulating protein-protein interactions.
There has been much interest in protein-protein interactions in the field of proteomics. A number of biochemical approaches have been used to identify interacting proteins. These approaches generally employ the affinities between interacting proteins to isolate proteins in a bound state. Examples of such methods include coimmunoprecipitation and copurification, optionally combined with cross-linking to stabilize the binding. Identities of the isolated protein interacting partners can be characterized by, e.g., mass spectrometry. See e.g., Rout et al., J. Cell. Biol., 148:635-651 (2000); Houry et al., Nature, 402:147-154 (1999); Winter et al., Curr. Biol., 7:517-529 (1997). A popular approach useful in large-scale screening is the phage display method, in which filamentous bacteriophage particles are made by recombinant DNA technologies to express a peptide or protein of interest fused to a capsid or coat protein of the bacteriophage. A whole library of peptides or proteins of interest can be expressed and a bait protein can be used to screening the library to identify peptides or proteins capable of binding to the bait protein. See e.g., U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,698; and 5,837,500. Notably, the phage display method only identifies those proteins capable of interacting in an in vitro environment, while the coimmunoprecipitation and copurification methods are not amenable to high throughput screening.
The yeast two-hybrid system is a genetic method that overcomes certain shortcomings of the above approaches. The yeast two-hybrid system has proven to be a powerful method for the discovery of specific protein interactions in vivo. See generally, Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, N.Y., 1997. The yeast two-hybrid technique is based on the fact that the DNA-binding domain and the transcriptional activation domain of a transcriptional activator contained in different fusion proteins can still activate gene transcription when they are brought into proximity to each other. As shown in FIG. 1, in a yeast two-hybrid system, two fusion proteins are expressed in yeast cells. One has a DNA-binding domain of a transcriptional activator fused to a test protein. The other, on the other hand, includes a transcriptional activating domain of the transcriptional activator fused to another test protein. If the two test proteins interact with each other in vivo, the two domains of the transcriptional activator are brought together reconstituting the transcriptional activator and activating a reporter gene controlled by the transcriptional activator. See, e.g., U.S. Pat. No. 5,283,173.
Because of its simplicity, efficiency and reliability, the yeast two-hybrid system has gained tremendous popularity in many areas of research. Numerous protein-protein interactions have been identified using the yeast two-hybrid system. The identified proteins have contributed significantly to the understanding of many signal transduction pathways and other biological processes. For example, the yeast two-hybrid system has been successfully employed in identifying a large number of novel cell cycle regulators that are important in complex cell cycle regulations. Using known proteins that are important in cell cycle regulation as baits, other proteins involved in cell cycle control were identified by virtue of their ability to interact with the baits. See generally, Hannon et al., in The Yeast Two-Hybrid System, Bartel and Fields, eds., pages 183-196, Oxford University Press, New York, N.Y., 1997. Examples of cell cycle regulators identified by the yeast two-hybrid system include CDK4/CDK6 inhibitors (e.g., p16, p15, p18 and p19), Rb family members (e.g., p130), Rb phosphatase (e.g., PP1-xcex12), Rb-binding transcription factors (e.g., E2F-4 and E2F-5), General CDK inhibitors (e.g., p21 and p27), CAK cyclin (e.g., cyclin H), and CDK Thr161 phosphatase (e.g., KAP and CDI1). See id. xe2x80x9c[T]he two-hybrid approach promises to be a useful tool in our ongoing quest for new pieces of the cell cycle puzzle.xe2x80x9d See id at page 193. In another example, the yeast two-hybrid system proved to be a powerful approach in analyzing the yeast pheromone response pathway, a complex multistep signal transduction process in haploid yeast cell mating. See generally, Sprague et al., in The Yeast Two-Hybrid System, Bartel and Fields, eds., pages 173-182, Oxford University Press, New York, N.Y., 1997. As described in Sprague, various genes were isolated from mutant yeast strains having altered pheromone response patterns. However, it was not clear how the proteins encoded by these genes function in the pheromone response pathway. The yeast two-hybrid system was utilized to test such proteins and mutant forms thereof for their ability to interact with each other. As a result, new insights and better understandings of the complex process were achieved. See id.
Reverse two-hybrid systems have also been developed based the yeast two-hybrid systems and are disclosed in, e.g., U.S. Pat. Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10315-10320 (1996); and Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10321-10326 (1996). Such reverse two-hybrid systems can be useful in identifying and selecting compounds capable of modulating a particular protein-protein interaction.
However, such reverse two-hybrid systems in the art typically depend on gene activation in the nucleus of host cells and has generally required that specific protein-protein interactions between fusion proteins occur within the nucleus of host cells. Thus, although certain conventional reverse yeast two-hybrid systems have been used successfully in identifying compounds capable of modulating protein-protein interactions, their usefulness may be limited when it is used in the context of protein-protein interactions that require non-nuclear environment. For example, traditional transcription-based reverse two-hybrid systems are not applicable to interactions between transcriptional factors. In addition, many cell surface proteins and their ligands contain disulfide bonds, which can be disrupted under the intracellular reducing conditions. Additionally, posttranslational protein modifications, particularly glycosylation, typically would preclude the nuclear localization of the modified proteins.
Cytosolic and cell surface protein-protein interactions play major roles in normal cellular functions and biological responses. In particular, many cytosolic and cell surface protein-protein interactions are involved in disease pathways. For example, attacks by pathogens such as viruses and bacteria on mammalian cells typically begin with interactions between viral or bacterial proteins and mammalian cell surface proteins. In addition, many protein-protein interactions between factors in the transcriptional machineries are also valuable drug targets. Therefore, there is a need in the art for improved methods that can be used to identify compounds capable of modulating such protein-protein interactions.
This invention provides a versatile and sensitive assay system for detecting protein-protein interactions that circumvents the above-described limitations inherent in prior art methods. Particularly, the present invention utilizes the so-called inteins, which are peptide sequences capable of directing protein trans-splicing both in vivo and in vitro. An intein is an intervening protein sequence in a protein precursor that is excised from the protein precursor during protein splicing. Protein splicing results in the concomitant ligation of the flanking protein fragments, i.e., the exteins, with a native peptide bond, thus forming a mature extein protein and the free intein. It is now known that inteins incorporated into non-native precursors can also cause protein-splicing and excision of the inteins. In addition, an N-terminal intein fragment in a fusion protein and a C-terminal intein fragment in another fusion protein, when brought into contact with each other, can bring about trans-splicing between the two fusion proteins. Thus, in accordance with the present invention, two hybrid fusion constructs are provided. One has a first test agent and an N-terminal intein fragment or N-intein, and the other has a second test agent and a C-terminal intein fragment or C-intein. In addition, one or both fusion constructs may have a reporter that undergoes detectable changes upon trans-splicing of the fusion constructs. If the first and second test agents interact with each other, thus bringing the N-intein and C-intein to close proximity, protein trans-splicing takes place. As a result, the fusion constructs are spliced, causing detectable changes in the reporter. Thus, by detecting the changes in the reporter, interactions between two test agents can be determined.
Intein-based trans-splicing can take place in vitro in a cell free environment. Therefore, the assay system of the present invention can be used for convenient and speedy in vitro analysis of protein-protein interactions. Particularly, the system can be easily adapted to high-throughput screening procedures.
In addition, trans-splicing can also occur inside a host cell in many different cellular backgrounds and compartments. In particular, unlike the traditional two-hybrid systems, the interacting proteins need not be transported into the cell nucleus. Thus, the system is useful in determining protein-protein interactions that require a specific cellular environment. For example, the system can be employed to detect interactions between nuclear proteins, between cytosolic proteins, and between membrane or extracellular proteins.
Additionally, protein trans-splicing mediated by the N-intein and C-intein is independent of other cellular factors and does not require the action of additional proteins such as proteases. This makes the assay system of the present invention more reliable and easier to perform as compared to the assay methods known in the art for detecting protein-protein interactions.
Another distinct feature of the intein-based assay is that the detection of protein-protein interaction is based on the occurrence of protein trans-splicing events, which typically are associated with protein cleavage and result in new protein structures and functions. Thus, the intein-based assay is well-suited to exploit the numerous direct and indirect methods available in the art for detecting changes in protein structures and functions. Because the intein-based assay can accommodate these numerous detection methods, there is great flexibility in choosing methods that are optimal for a particular condition.
Accordingly, in accordance with a first aspect of the present invention, a method for detecting protein-protein interaction is provided. Briefly, two fusion proteins are prepared and allowed to interact with each other. One of the two fusion proteins includes an N-intein and a first test polypeptide, and the other fusion protein includes a C-intein and a second test polypeptide. One or both of the two fusion proteins have an inactive reporter capable of being converted to an active reporter upon trans-splicing through the N-intein and the C-intein. The change in the active reporter level is determined. An increase in the amount of the active reporter would indicate that the first and second test polypeptides interact with each other through, e.g., binding affinity, to result in the trans-splicing of the two fusion proteins mediated by the N-intein and the C-intein. Preferably, the N-intein and C-intein are not associated with each other and do not exhibit any significant binding affinity to each other. Nor do they associate with or bind to the inactive reporter or test polypeptides in the fusion proteins.
In one embodiment, the inactive reporter can be a polypeptide linked to one of the fusion proteins, and is cleaved off into a free form from the fusion protein upon protein trans-splicing. The reporter polypeptide can be selected and the fusion proteins can be designed such that the precursor form of the polypeptide is inactive while the free reporter released from the fusion protein is active, i.e., is detectable directly or indirectly.
In another embodiment, one of the two fusion proteins has a nonfunctional portion of a reporter polypeptide linked to the N-terminus of the N-intein. The other fusion protein comprises a distinct but similarly nonfunctional portion of the same reporter polypeptide linked to the C-terminus of the C-intein. Upon trans-splicing between the two fusion proteins through the N- and C-inteins, the two inactive reporter polypeptides are ligated together with a peptide bond, thereby forming an active reporter protein, which is detectable directly or indirectly.
The assay can be conducted in vitro in a substantially cell free environment by mixing together purified forms of the two fusion proteins under conditions suitable for protein interactions and for protein trans-splicing. Alternatively, the fusion proteins can be recombinantly expressed separately in different host cells, and cell lysates or crude extracts prepared from the cells can be mixed to allow protein-protein interaction. The active reporter protein is then detected.
The assay can also be conducted in vivo by allowing the fusion proteins to interact within a host cell. Suitable cells include, but are not limited to, bacteria cells, yeast cells, plant cells, insect cells and animal cells. Chimeric genes encoding the above-described fusion proteins are introduced into a host cell to express recombinantly the fusion proteins. The amount of the active reporter protein in the host cell is determined. In one embodiment, a first chimeric gene encoding one of the two fusion proteins is expressed in a haploid Saccharomyces cell of a mating type and a second chimeric gene encoding the other fusion protein is expressed in a haploid Saccharomyces cell of mating type xcex1. The two cells are mated to form a diploid cell, and any change in the amount of the active reporter protein in the diploid is then determined.
In a specific embodiment of the in vivo assay, expression of one or more of the chimeric genes can be made inducible, e.g., by placing the genes under control of an inducible promoter, such that one or more of the fusion proteins are produced when the host cell is subject to a predetermined condition.
In yet another embodiment of the in vivo assay, the fusion proteins can have a signal peptide and optionally a membrane anchoring domain such that the fusion proteins recombinantly expressed in the host cells are secreted extracellularly or anchored on cell surface.
In addition, the assay can also be conducted in the presence of a third polypeptide. In this manner, the interaction between the first and second test polypeptides can be detected if the interaction requires the presence of the third polypeptide. The third polypeptide may be a protein having affinity to either the first or second test polypeptides or both. Alternatively, the third polypeptide can modify one or both test polypeptides, e.g., by phosphorylation, glycosylation, and the like.
The techniques used for monitoring the occurrence of protein trans-splicing events and detecting an active reporter will depend on the inactive reporter used and the active reporter derived therefrom. The system of the present invention can be designed such that an active reporter can be detected based on changes in protein sizes or other properties, or activation of certain protein functions. For example, in an in vivo system, detection of an active reporter can be based on cell viability assays, color assays, and the like.
In accordance with a second aspect of the present invention, the above-described assay system is employed to determine whether a compound is capable of interfering with an interaction between a first polypeptide and a second polypeptide. Essentially, two fusion proteins as described above are provided except that the first and second polypeptides are known to interact with each other. The interaction between the two fusion proteins in the presence of the test compound is determined. Many suitable reporters can be used in this screening assay system. Preferably, a reverse or negative selection technique is incorporated into the screening assay of the present invention. For example, the fusion proteins can be designed such that the active reporter generated by protein trans-splicing is toxic to the host cell or represses the expression of a detectable gene. In this manner, compounds capable of interfering with the interaction between the two fusion proteins can be identified based on, e.g., the survival of the host cell or the expression of a detectable gene.
In accordance with another aspect of the present invention, a composition for detecting protein-protein interactions is provided, which includes a first chimeric gene encoding a first fusion protein and a second chimeric gene encoding a second fusion protein. The first fusion protein includes an N-intein and a first test polypeptide. The second fusion protein has a C-intein and a second test polypeptide. One or both of the fusion proteins has an inactive reporter protein capable of being converted to an active reporter protein upon trans-splicing through the N-intein and the C-intein. In one embodiment, each of the chimeric genes is contained in an expression vector, respectively. The expression vectors also contain elements necessary for the replication of the vector in a host cell, the correct transcription and translation of the chimeric genes (e.g., promoters and other transcriptional regulatory elements, transcription termination signal, etc.). The vectors preferably also contain a selection marker gene for selecting those host cells harboring the expression vectors. Preferably, the N-intein and the C-intein are incapable of interacting with each other or with the inactive reporter.
The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples and drawings, which illustrate preferred or exemplary embodiments.